Effects of polymyxin B on the sarcoplasmic reticulum membrane

1989 ◽  
Vol 9 (5) ◽  
pp. 573-578 ◽  
Author(s):  
T. B. Ktenas ◽  
T. G. Sotiroudis ◽  
A. E. Evangelopoulos
Keyword(s):  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Fluorescence Intensity ◽  
Cyclic Peptide ◽  
Fluorescein Isothiocyanate ◽  
Polymyxin B ◽  
The Other ◽  
Peptide Antibiotic ◽  
Phosphorylase Kinase ◽  
Sarcoplasmic Reticulum Membrane

Polymyxin B, a cyclic peptide antibiotic, inhibits Ca2+-ATPase, p-nitrophenyl phosphatase and phosphorylase kinase activities associated with rabbit skeletal muscle sarcoplasmic reticulum membranes; 50% inhibition is induced by 100 μM, 130μM and 550 μM of polymyxin respectively. The fluorescence intensity of fluorescein isothiocyanate-labeled Ca2+-ATPase, decreases in the presence of polymyxin (50% of the total decrease at 70 μM polymyxin). On the other hand, the polypeptide inhibits calmodulin-dependent endogenous phosphorylation of 60 kDa, 20 kDa and 14 kDa membrane proteins, while an increase of calmodulin-dependent phosphorylation is observed in 132 kDa and 86 kDa proteins.

scholarly journals A model for the phosphorylation of the Ca2+ + Mg2+-activated ATPase by phosphate

1986 ◽  
Vol 237 (1) ◽  
pp. 207-215 ◽  
Author(s):  
R J Froud ◽  
A G Lee
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Fluorescence Intensity ◽  
Fluorescein Isothiocyanate ◽  
Fluorescence Response

We have shown that changes in fluorescence intensity for the Ca2+ + Mg2+-activated ATPase of sarcoplasmic reticulum labelled with fluorescein isothiocyanate following the addition of Ca2+ can give the ratio of the two conformations (E1 and E2) of the ATPase. We show that the fluorescence response to Ca2+ is unaffected by Mg2+, phosphate or K+, implying that these ions bind equally well to the E1 and E2 conformations. A model is presented for phosphorylation of the ATPase by phosphate as a function of pH, Mg2+, K+ and Ca2+.

scholarly journals Stereorandomization as a Method to Probe Peptide Bioactivity

2020 ◽  
Author(s):  
Thissa N. Siriwardena ◽  
Bee-Ha Gan ◽  
Thilo Köhler ◽  
Christian van Delden ◽  
Sacha Javor ◽  
...  
Keyword(s):  
Cyclic Peptide ◽  
Solid Phase ◽  
Solid Phase Peptide Synthesis ◽  
Therapeutic Index ◽  
Building Blocks ◽  
Polymyxin B ◽  
High Yield ◽  
Peptide Antibiotic ◽  
Disruptive Effect ◽  
Peptide Dendrimers

<p>Solid-phase peptide synthesis (SPPS) is usually performed with optically pure building blocks to prepare peptides as single enantiomers. Herein we report that SPPS using racemic amino acids provides stereorandomized (<i>sr</i>) peptides, containing up to billions of different stereoisomers,<a> </a>as well-defined single HPLC peak, single mass products with high yield, which can be used to investigate peptide bioactivity. To exemplify our method, we show that stereorandomization abolishes the membrane disruptive effect of α-helical amphiphilic antimicrobial peptides but preserves their antibiofilm effect, implying different mechanisms involving folded versus disordered conformations. For antimicrobial peptide dendrimers by contrast, stereorandomization preserves antibacterial, membrane disruptive and anti-biofilm effects but reduces hemolysis and cytotoxicity, thereby increasing their therapeutic index. Finally, we identify partially stereorandomized analogs of the last resort cyclic peptide antibiotic polymyxin B with preserved antibacterial activity but lacking membrane disruptive and lipopolysaccharide neutralizing activity, pointing to the existence of additional targets.</p>

Calcium removal kinetics of the sarcoplasmic reticulum ATPase in skeletal muscle

1997 ◽  
Vol 272 (4) ◽  
pp. C1087-C1098 ◽  
Author(s):  
E. E. Burmeister Getz ◽  
S. L. Lehman
Keyword(s):  
Skeletal Muscle ◽  
Steady State ◽  
Sarcoplasmic Reticulum ◽  
Initial Rate ◽  
Troponin C ◽  
The Other ◽  
Physiological Conditions ◽  
Initial Binding ◽  
Kinetics Of ◽  
Transient And Steady State

The models of the sarcoplasmic reticulum (SR) Ca pump used to simulate Ca kinetics in muscle fibers are simple but inconsistent with data on Ca binding or steady-state uptake. We develop a model of the SR pump that is consistent with data on transient and steady-state Ca removal and has rate constants identified under near-physiological conditions. We also develop models of the other main Ca-binding proteins in skeletal muscle: troponin C and parvalbumin. These models are used to simulate Ca transients in cut fibers during and after depolarizing pulses. Simulations using the full SR pump model are contrasted with simulations using a Michaelis-Menten (MM) approximation to SR pump kinetics. The MM pump underestimates the amount of Ca released during depolarization, underestimates the initial rate of Ca binding by the pump, and overestimates the later rate of Ca pumping. These errors are due to fast initial binding by the SR pump, which is neglected in the MM approximation.

scholarly journals Effects of ATP on the Interaction of Ca++, Mg++, and K+ with Fragmented Sarcoplasmic Reticulum Isolated from Rabbit Skeletal Muscle

1967 ◽  
Vol 50 (5) ◽  
pp. 1327-1352 ◽  
Author(s):  
Arselio P. Carvalho ◽  
Barbara Leo
Keyword(s):  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Binding Sites ◽  
Membrane Fraction ◽  
Binding Capacity ◽  
Cation Binding ◽  
The Other ◽  
Surface Binding ◽  
Ph Values ◽  
Number Of Binding Sites

Fragmented sarcoplasmic reticulum isolated from skeletal muscle of the rabbit has a cation-binding capacity of about 350 µeq/g of protein at neutral pH. The same binding sites bind Ca, Mg, K, and H ions and, consequently, the selective binding of Ca induced by ATP releases an amount of the other cations equivalent to the Ca taken up. At pH values below 6.2, an increasing number of binding sites are associated with H+, and ATP induces exchange of Ca mostly for H+. At pH values above 6.2, the binding sites exist in the form of Mg and K, and Ca is bound in exchange for these cations. The total bound Ca + Mg + K, expressed in microequivalents of cations bound per gram of protein, is approximately constant at various pCa values, which indicates a stoichiometric exchange of Ca for the other cations. To accomplish the same degree of exchange of Ca for other cations bound, in the absence of ATP, concentrations of free Ca++ of about 1000-fold higher than those needed in the presence of ATP are required in the medium. We cannot distinguish between a mechanism whereby Ca actively transported into a compartment of the microsomal vesicles containing also the binding sites is bound passively to these sites in exchange for Mg, K, and H and another in which ATP selectively increases the affinity of surface-binding sites for Ca. Irrespective of the mechanism of accumulation, the Ca retained does not contribute to the activity of the cation in the membrane fraction. Caffeine (10 mM) has no effect on the binding of Ca, but releases a more labile fraction of Ca, which presumably accumulates in excess of the bound Ca. Procaine (5 mM) antagonizes the effect of caffeine. Acetylcholine and epinephrine have no effect on the binding of Ca.

scholarly journals The effects of membrane potential and lanthanides on the conformation of the Ca2+-transport ATPase in sarcoplasmic reticulum

1986 ◽  
Vol 234 (2) ◽  
pp. 363-371 ◽  
Author(s):  
I Jona ◽  
A Martonosi
Keyword(s):  
Membrane Potential ◽  
Sarcoplasmic Reticulum ◽  
Fluorescence Intensity ◽  
Binding Sites ◽  
Fluorescein Isothiocyanate ◽  
Tryptophan Fluorescence ◽  
Lanthanide Ions ◽  
Positive Potential ◽  
High Affinity ◽  
Ion Substitution

The effects of Ca2+, lanthanide ions (Gd3+, La3+ and Pr3+) and membrane potential on the fluorescence of tryptophan and covalently bound fluorescein were analysed in native and fluorescein isothiocyanate (FITC)-labelled sarcoplasmic reticulum vesicles. The binding of Ca2+ and lanthanides to the Ca2+-ATPase increases the fluorescence intensity of tryptophan and decreases the fluorescence intensity of FITC; the dependence of these effects on cation concentration is consistent with the involvement of the high-affinity Ca2+-binding sites of the Ca2+-ATPase in the cation-induced fluorescence changes. The fluorescence of FITC-labelled sarcoplasmic reticulum vesicles is also influenced by membrane potential changes induced by ion substitution. Inside positive potential increases, while inside negative potential decreases, the fluorescence of bound FITC. Smaller potential-dependent changes in tryptophan fluorescence were also observed. The effects of Ca2+, lanthanides and membrane potential on the fluorescence of tryptophan and FITC are discussed in terms of the two major conformations of the Ca2+-ATPase (E1 and E2), that are assumed to alternate during Ca2+ transport. The observations support the suggestion [Dux, Taylor, Ting-Beall & Martonosi (1985) J. Biol. Chem. 260, 11730-11743] that the vanadate-induced crystals of Ca2+-ATPase represent the E2, while the Ca2+ and lanthanide-induced crystals the E1, conformation of the enzyme.

scholarly journals Effects of pH on phosphorylation of the Ca2+-ATPase of sarcoplasmic reticulum by inorganic phosphate

1997 ◽  
Vol 321 (3) ◽  
pp. 671-676 ◽  
Author(s):  
Yamin M. KHAN ◽  
J. Malcolm EAST ◽  
Anthony G. LEE
Keyword(s):  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Equilibrium Constant ◽  
Fluorescence Intensity ◽  
Inorganic Phosphate ◽  
Ph Dependence ◽  
Phosphorylated Form ◽  
Phosphorylation Reaction ◽  
Effects Of Ph ◽  
High Concentrations

The fluorescence intensity of the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum (SR) labelled with 4-(bromomethyl)-6,7-dimethoxycoumarin has been shown to decrease on phosphorylation of the ATPase with Pi, this providing a convenient measure of the level of phosphorylation. Comparison of the fluorescence decrease observed with ATP and with high concentrations of Pi fix the value of the equilibrium constant for the phosphorylation reaction E2PMg ⇌ E2PiMg at pH 6.0 at about 2. Studies of the pH-dependence of phosphorylation show that H2PO4- and HPO42- bind to the ATPase with equal affinity, but that only binding of H2PO4- leads to phosphorylation, described by an equilibrium constant of 2.3. Luminal Ca2+ can bind to a pair of sites on the ATPase, with affinities of 1.3ȕ103 and 1.7ȕ103 M-1 for the unphosphorylated and phosphorylated forms of the ATPase respectively, with stronger binding of Ca2+ to the phosphorylated form resulting in an increase in the effective equilibrium constant for phosphorylation.

scholarly journals Conformational transitions in the Ca2+ + Mg2+-activated ATPase and the binding of Ca2+ ions

1986 ◽  
Vol 237 (1) ◽  
pp. 197-206 ◽  
Author(s):  
R J Froud ◽  
A G Lee
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Fluorescein Isothiocyanate ◽  
Kinetic Studies ◽  
Conformational Transitions ◽  
The Other ◽  
Effect Of Ph ◽  
High Affinity

We have studied the fluorescence of the Ca2+ + Mg2+-activated ATPase of sarcoplasmic reticulum labelled with fluorescein isothiocyanate. The change in intensity of fluorescein fluorescence caused by addition of Ca2+ to the labelled ATPase can be interpreted in terms of a two-conformation model for the ATPase, one conformation (E1) having a high affinity for Ca2+, the other (E2) a low affinity. Effects of Ca2+ as a function of pH allow an estimate of the effect of pH on the E1/E2 ratio, consistent with kinetic studies. A model is presented for binding of Ca2+ to the ATPase as a function of pH that is consistent both with the data on the E1/E2 equilibrium and with literature data on Ca2+ binding.

Is Carbonic anhydrase Required for Contraction of Skeletal Muscle?

Physiology ◽  
1991 ◽  
Vol 6 (2) ◽  
pp. 78-82
Author(s):  
C Geers ◽  
P Wetzel ◽  
G Gros
Keyword(s):  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Carbonic Anhydrase ◽  
Sarcoplasmic Reticulum Membrane ◽  
Simultaneous Transfer

An important step in excitation-contraction doupling is release and reuptake of Ca2+ at the sarcoplasmic reticulum membrane, which requires simultaneous transfer of counterions, probably H+. Evidence is provided that a carbonic anhydrase associated with the sarcoplasmic reticulum membrane rapidly generates the H+ necessary for this exchange.

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